Isolation of human DNA-unwinding elements as sites of DNA polymerase α/primase entry

Robert A. Pack, Toshiki Tsurimoto

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

Human DNA libraries were screened for DNA synthesis activity in vitro using purified DNA polymerase α/primase and a viral DNA helicase (simian virus 40 large tumor antigen). Three clones exhibited a high activity distinguishable from the rest. The DNA synthesis was dependent on negative supertwisting and initiated at a unique region in the human DNA insert. Functional subclone DNA fragments which could be shortened to less than 1 kb are located in the initiation region. Binding with a single-stranded DNA-binding protein and digestion with nuclease P1 demonstrated that these DNAs have a highly single-stranded nature at a certain site in a closed circular plasmid. The minimal functional sequences coincide with the single-stranded region and contain a characteristic dinucleotide repeat sequence. These repeats have an extremely low free energy for DNA strand separation and are denned as DNA-unwinding elements, which are frequently observed at regions flanking replication origins in Escherichia coli and Saccharomyces cerevisiae chromosomes. We suggest that such a repeating sequence would have an important role during initiation of DNA replication and function as a site to recruit replication proteins. DNA replication; DNA helicase; dinucleotide repeats; replication protein A; nuclease P1.

Original languageEnglish
Pages (from-to)277-284
Number of pages8
JournalGene
Volume148
Issue number2
DOIs
Publication statusPublished - Oct 21 1994
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Genetics

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